CN102844328A - 胰岛素样肽 - Google Patents
胰岛素样肽 Download PDFInfo
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- CN102844328A CN102844328A CN2010800534723A CN201080053472A CN102844328A CN 102844328 A CN102844328 A CN 102844328A CN 2010800534723 A CN2010800534723 A CN 2010800534723A CN 201080053472 A CN201080053472 A CN 201080053472A CN 102844328 A CN102844328 A CN 102844328A
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- Prior art keywords
- chain
- peptide
- insulin
- polypeptide
- disulfide linkage
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Abstract
描述了通过随机结合A链与B链制备胰岛素样肽、嵌合型胰岛素样肽及其衍生物的方法,和所得产物的药学应用。
Description
胰岛素样肽(INSL),例如结构类似于胰岛素的这些肽,由2条肽链:A链和B链组成,此2条链通过2个分子间二硫键连接在一起,同时A链含有一个额外的分子内二硫键。一个例外是***IGF-1和IGF-2,它们各由含70个氨基酸的一条肽链组成。
INSL家族除胰岛素外还包括INSL3、INSL4、INSL5、INSL6、松弛素1(RLN1)、松弛素2(RLN2)和松弛素3(RLN3)以及生长因子IGF-1和IGF-2。
INSL肽显示具有重要的生物学性能,它们决定了代谢,例如胰岛素和IGF-1,并能调节生物体的重要状态,例如妊娠受RLN1、RNL-2和INSL4调节。
胰岛素销售用于治疗糖尿病并可视为最佳的被研究蛋白质,IGF-1用于重度原发性IGF-1缺陷病例治疗并在多项临床试验中针对适应征例如I型糖尿病、II型糖尿病、阿尔茨海默症、严重烧伤和肌强直性肌营养不良症(MMD)等,RLN2在临床试验中测试了对急性心力衰减、先兆子痫和硬皮病的效果,除此以外关于其他INSL及它们的衍生物和拮抗物的生物学性能和可能的治疗应用知之甚少。
一种情况中表明由一种INSL的A链和另一种INSL的B链组成的嵌合肽能与相应INSL的独特受体相互作用并显示显著的生物学活性。此外,已表明由IGF-1的A部分和IGF-2的B部分组成肽链的IGF具有额外的重要生物学活性。对于大量的其它嵌合肽证明同样如此。
尚未深入研究嵌合性INSL的生物学性能和可能的药学应用,因为它们的制备非常困难。事实上只制备和研究了由INSL5的B链和其它INSL的各种A链组成的嵌合肽。特别是,已发现由RLN3的A链和INSL5的B链组成的RLN3A/TNSL5与GPCR135和GPCR142受体相互作用。
对于由INSL的二条不同链组成的嵌合肽仅有限研究的原因是其合成的难度和低产率。
迄今已采用的方法是A)随机混合直链A链与B链及其氧化,B)混合在半胱氨酸残基的巯基处含磺酸基团的A链,和C)定点构建A链与B链之间的三个二硫键。认为此方法是迄今为止制备INSL的最为改进的化学方法,虽然它需要多个步骤和5步层析纯化。显然所有上述方法非但不尽人意而且导致制备少量INSL的难度大及成本高。此外,这些方法不适合大规模生产。
由于化学合成困难,故采用重组DNA技术生产这些肽,例如胰岛素、松弛素和IGF。但是重组DNA技术比常规的肽和蛋白质合成方法更为复杂。因此,即使在最简单的IGF-1的情况中,还需要在分离得到其直链后选择性形成3个二硫键。
产生由2条肽链组成的INSL变得甚至更为困难,因为该情况中用重组DNA技术合它们的前肽,如前胰岛素、前松弛素等前肽后,需要选择性形成二硫键并最后用酶去除各前肽中间的C-肽段。
制备RLN2甚至更为复杂,在切除C肽段后需要额外的步骤通过加热将位于A链氨基末端的谷氨酰胺残基转变为焦谷氨酸残基。因此,即使生物技术制备的INSL也很难制成,且需要多步层析纯化才能获得药学上可接受纯度的INSL。这些巨大的困难和高生产成本导致极大地延误了对许多INSL生物学性能的评价和在临床试验中对它们的药学应用测试,虽然认为它们的生物学活性是肯定的。这对于含胰岛素链与其他INSL链结合的嵌合性INSL同样成立。
发明内容
近年来我们发现并报道了一种随机重组RLN1和RLN2链的简单制备方法。该方法显示很容易合成嵌合肽,例如由RNL2A/RLN1B所组成。用单环A链和双环A链时该合成方法特别有效。这表明A链含有结构信息使其能与不对应链的天然对的胰岛素B肽链结合。
在本发明中,我们描述了胰岛素样肽A链的结构特征使它识别并选择性结合胰岛素的所有B链和其它与那些肽相类似的链。肽链的结合总是选择性给出对应预期和天然胰岛素样的结合。
例如,本发明表明双环RLN2A识别并通过随机组合不仅连接其它INSL的B链,还与理论上对应于INSL的B链和对应于作为单链肽而天然所见的IGF-1和IGF-2的B链随机结合。这同样可用于其它胰岛素样肽的结合,使我们有可能容易地制备具有可能的药学活性的嵌合多肽。
在本发明中,描述了单环A链和双环A链反应的主要副产物是B链氧化成环形B链。具体说,INSL的直链B链与双环A链的反应非常快,如果A链过量在数分钟内即可将该B链氧化成环形B链同步形成胰岛素样肽。
双环A链肽有效氧化和产生二硫键连接的这种性能是一种氧化酶性能,因此我们可将胰岛素样肽的双环A链称为已知的最小的同时为强效的氧化酶,其还显示能容易地组合其它肽链的能力。
正是这种性能使得INSL的双环A链和类似的肽链作为用于蛋白质构象疾病的感兴趣治疗剂。因此,提供合适的双环INSL A链或类似肽,可能有助于改善因生物体功能障碍导致的蛋白质折叠缓慢。
我们还揭示INSL的双环A链易与只含不参与二硫键的半胱氨酸残基(例如导致糖尿病的某些胰岛素突变体中)的肽链反应。
为此,给予双环A链作为药物可对于通过它与突变蛋白的结合反应,然后通过生物体的ERAD***或其它防御***,破坏双环A链与突变蛋白的这种结合,来清除人体或动物体中的突变蛋白极其有益。
双环A链作为药物的递送将极为有益,因为它会与沉淀蛋白反应并溶解它们,还能与蛋白寡聚物或多聚物反应而溶解它们,并通过其氧化活性使之折叠而恢复它们的功能。
在本发明的另一种实施方式中,我们描述了一种INSL肽链的方便而有效的合成方法,通过采用2-氯三苯甲基和4-甲基二苯甲基化树脂的固相合成技术。此外,可采用本领域已知用于制备直链肽或肽酰胺的所有技术。
本发明描述了已知的INSL和嵌合肽的改进的化学合成方法。还首次描述了由2条链组成且链以胰岛素与其它胰岛素样肽2条链的链连接方式连接的IGF-1和IGF-2的嵌合性衍生物(图1),及。还首次描述了由一种INSL的A链与另一种INSL的B链组成的一系列嵌合性INSL。
正确形成二硫键对INSL的肽合成非常重要。在本发明中,我们描述了形成正确-S-S-结合的方法。半胱氨酸残基的这些氧化反应可在各个链纯化之前或之后进行。也可用带保护形式的肽形成二硫化物/键连接。
如果2条链的合成采用固相法,二硫键的形成可以在树脂上、从树脂上切割肽后或从树脂上切割肽的同时进行。氧化半胱氨酸巯基以形成分子内二硫键可采用任何氧化剂进行,但优选用二甲基亚砜(DMSO)(JP Tam等,J Am ChemSoc,1991,113:6657-6662)氧化去保护INSL链;带保护的肽或部分去保护的肽则用碘氧化。
链装配时为了保护半胱氨酸的侧链巯基,可利用科学领域已知的保护巯基功能团的各种保护基团,但优选利用4-甲氧基三苯甲基(Mmt)(Barlos等,Int.J.Peptide Protein Res.1996,47,148-153)、三苯甲基(Trt)和乙酰胺基甲基(Acm)基团。
在本发明中,我们也描述了通过将胰岛素样肽的A链和B链氧化成相应的双环和单环A链和B链而提高其溶解度。因此在制备型高效液相层析(HPLC)中它们的洗脱大大早于相应的(还原)肽,因而对于纯化它们的应用简单而优于直链(还原)肽的应用。
为了在A链中选择性形成分子间二硫键,可利用任何一对正交保护基团,但优选采用Trt/Mmt、Trt/Acm和Mmt/Acm三对中的一对。
当采用Trt/Mmt对时,选择性去除S-Mmt基团后用合适的氧化剂,优选用空气或DMSO使已释放的游离巯基之间形成二硫键。优选用碘氧化去除S-Trt和Acm基团形成第2个二硫键。采用2-氯三苯甲基树脂(K.Barlos等,Int.J.Pept.Protein Res.1991,37,513-520)或对酸具有相似敏感性的树脂固相合成A链时,用酸温和切割选择性去除S-Mmt基团与从树脂上切割该肽同时进行。
对于氧化去除S-Trt基团然后形成二硫键,可采用该领域已知的任何氧化剂但优选采用碘。
如果利用Trt/Acm对,可利用合适的酸溶液,优选用含10-100%浓度三氟乙酸的二氯甲烷液对该肽树脂进行酸处理,并以不同比例加入清除剂优选硫醇,甲硅烷和水,在S-Acm基团存在下选择性去除S-Trt基团。第1个二硫键的形成可采用该领域已知的任何氧化剂实现,但优选DMSO和空气。
可用碘氧化去除S-Trt基团形成第1个二硫键,这可在将带保护的肽从树脂上切割之前、切割时或切割后进行(K.Barlos等,Int.J.of Peptide&Protein Research,1991,38,562-568)。
若在0°C-15°C低温下于亲脂溶剂,优选氯化烃、氟化醇中,用温和的酸如乙酸和三氟乙酸进行碘化反应,可在S-Acm基团存在下选择性形成所需的二硫键。
通过加入极性组分如乙酸、甲醇、三氟乙酸和偶尔用水,可在更极性的溶剂中产生第2个二硫键。碘化期间的温度可以不同但优选设置在5-25°C范围内。
胰岛素样肽的固相合成可采用科学领域已知的任何树脂进行,但优选三苯甲基类型的树脂,如2-氯三苯甲基树脂(K.Barlos等,Tetrahedron Lett.,1989,30,3943;K.Barlos等,Tetrahedron Lett.,1989,30,3947;K.Barlos等,Angew.Chem.Int.Ed.Engl.,1991,30,590;K.Barlos等,Int.J.Pept.Protein Res.,1991,37,513;Barlos,等,Int.J.Pept.Protein Res.,1991,38,562)和4-甲基二苯甲基溴树脂(K.Barlos等,Liebigs Annalen der Chemie(1989),(10),951-5)。
在本发明中,我们描述了胰岛素样肽的A和B链结合(折叠)的改进方法(图3-9)。含有分子内二硫键的环形肽在形成分子间-SS-键时通常比相应的直链肽反应更快。它们表现为活化的环肽并以更有效的方式与第二条链进行分子间结合。在混有A链异构体和B链的混和物中用DMSO、空气或其它氧化剂氧化直链的肽。本发明描述了混有A链的多种双环异构体或单独异构体的化合物与环形B链(图6)催化反应产生所需产物。加入还原性催化剂加速该反应。催化剂将二硫桥还原成游离巯基,产生环形肽与分子间连接的肽之间的平衡。这导致后续反应产生热力学更稳定的产物即天然蛋白质。
所用的还原剂可以是任何有机或无机物质,但优选有机硫醇,例如还原(直)链A和/或B还原型谷胱甘肽、半胱氨酸、苯硫酚、吡啶硫醇、3或5硝基吡啶-2-硫醇、苄基硫醇(benzylmercaptane)、二硫苏糖醇等。优选采用A或B链或它们的混合物作为催化剂。可在A链与B链混合之前、混合期间或之后加入催化剂。
可加入不同量的催化剂以达到平衡。折叠期间的温度可以不同但优选设置在24°C。采用水或水与有机溶剂的混合液作溶剂,偶尔加入碱。链结合时的pH可以不同但优选设为pH 10-11。
在本发明中我们也表明存在合适的氧化剂如DMSO时,还原的A链能与B链结合(折叠)形成胰岛素样肽。该反应通过形成单环和双环A链的混合物而进行。
采用已氧化的A链与B链时链的结合更快。此时,混和的A链与B链反应来在所有情况中给出胰岛素样肽,具有其二硫键的物理排列。
优选通过加入15%DMSO作为氧化剂来进行双环A链与还原(直)的B链结合以完成折叠(图4)。A链与B链的比例可以不同但优选摩尔比1.1∶1。增加反应液中的A链过量可加速反应。此种情况下可在HPLC纯化胰岛素样肽时回收过量的A链或B链。
胰岛素样肽的纯化由HPLC用各种溶剂混合液进行,但优选含三氟乙酸(TFA)、甲酸或乙酸的水和乙腈中进行。可通过冻干或沉淀分离已纯化的胰岛素样肽。如果需要进行脱盐,可采用常用的强离子交换树脂,例如Dowex。
实施例
实施例1
固相合成胰岛素样肽的A链、B链和它们的带保护区段。通用程序。
A1.制备负载的2-氯三苯甲基树脂,通用程序。
取CBL-Patras公司的2-氯三苯甲基氯树脂(CTC-C1)(100g;载量1.6mmol/g)置于2L肽合成反应罐中,用700mL二氯甲烷(DCM)25°C溶胀30分钟。过滤树脂,加入含100mmol Fmoc-氨基酸和300mmol二异丙基乙胺(DIEA)的500mLDCM溶液。混合物于氮气下在25°C搅拌2小时。然后加入10mL甲醇(MeOH)反应1小时,中和2-CTC树脂的残留活性位点。过滤树脂并用400mL DMF洗涤2次。过滤树脂,用含25%体积哌啶的500mL DMF处理30分钟2次。然后用500mL DMF洗涤树脂4次。用500mL异丙醇(IPA)洗涤3次使树脂去溶胀。干燥树脂至恒重。所用毫摩尔的氨基酸70-95%结合在树脂上。
A2.制备负载的MBH-树脂,通用方法。
将MBH-Br树脂(100g;190mmol)置于2L肽合成仪中,用700mL DCM 25°C溶胀30分钟。过滤树脂,然后加入含Fmoc-氨基酸和DIEA的500mL DCM溶液。混合物在氮气下25°C搅拌6小时。然后加入10mL MeOH搅拌24小时以结合MBH树脂的残留活性位点。过滤树脂,用400mL DMF洗涤2次。过滤树脂,与含25%体积哌啶的500mL DMF溶液再反应30分钟2次。然后用500mL DMF洗涤树脂4次。用500mL IPA洗涤3次使树脂去溶胀。然后树脂在真空(15torr,25°C)下干燥至恒重。所用氨基酸中60-90%的豪摩尔数结合在树脂上。
B.固相合成,通用方案
固相合成用1.0g如实施例部分A中所述酯化于CTC或MBH树脂上的氨基酸在24°C下进行。整个合成中采用以下方案。
B1.溶胀树脂
将树脂置于15ml反应器中用7ml NMP处理2次,然后过滤。
B2.活化氨基酸
称取氨基酸(3.0当量)和1-羟基苯并***(4.0当量)置于装有2.5倍体积NMP的反应器中溶解并冷却至0°C。然后加入DIC(3.0当量)并将混合液搅拌15分钟。
B3.偶合
将B2制备的溶液加入到B1反应器中。在反应器中加入一倍体积的DCM洗涤反应器1次,在25°C-30°C搅拌1-3小时。取样做Kaiser试验确定反应是否完成。如果3小时后偶合反应未完成(Kaiser试验阳性),过滤反应混和液并与新鲜的活化氨基酸溶液再偶合。完成偶合后过滤反应混和液用NMP洗涤4次(每次洗涤用5倍体积)。
B4.去除Fmoc-基团
过滤B3得到的树脂用含25%体积哌啶的5mL溶液处理30分钟。然后用5mLNMP洗涤树脂3次。
B5.肽链延伸
重复步骤B1-B5,掺入各个氨基酸直至完成肽链。
为引入每一个氨基酸,采用了以下Fmoc-氨基酸:Fmoc-Gly-OH、Fmoc-Ala-OH、Fmoc-Val-OH、Fmoc-Ile-OH、Fmoc-Leu-OH、Fmoc-Met–OH、Fmoc-Met(O)-OH、Fmoc-Phe-OH、Fmoc-Pro-OH、Fmoc-Asp(tBu)-OH、Fmoc-Glu(fBu)-OH、pGlu、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Thr(Trt)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Tyr(Clt)-OH、Fmoc-Asn-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gln-OH、Fmoc-Gln(Trt)-OH、Fmoc-Trp-OH、Fmoc-Trp(Boc)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-His(Trt)-OH、Fmoc-Cys(Trt)-OH、Fmoc-Cys(Mmt)-OH和Fmoc-Cys(Acm)-OH,及以下Boc-氨基酸:Boc-Arg(Pbf)-OH、Boc-Gln-OH、Boc-Gln(Trt)-OH、Boc-Lys(Boc)-OH和Boc-Asp(tBu)-OH。
C.从CTC树脂上切割胰岛素样肽及其在N-末端含有Fmoc-或Boc-基团的带保护区段的通用方法。
上述B1-B5产生的结合有肽或肽区段的树脂用5mL NMP洗涤4次,用5mLIPA洗涤3次,最后用7mL DCM洗涤5次,以完全去除任何残留的NMP或其它碱性组分。然后冷却树脂至0°C,过滤去除DCM,用10mL 1%TFA/DCM液5°C处理2次。然后将混合液在0°C搅拌20分钟后过滤。树脂用10mL DCM洗涤3次。然后滤液中加入吡啶(相当于TFA的1.3当量)以中和TFA。然后将该DCM切割液与等体积水混和。所得混和液减压蒸馏(28°C下350torr)以去除DCM。去除DCM后沉淀肽或肽区段。然后用水洗涤所得肽并在30-35°C下15Torr真空干燥。
实施例2
胰岛素样肽的去保护。通用方法
将上述实施例1中所得带保护的A链和B链(0.01mmol)用10mL TFA/DTT/水(90∶5∶5)5°C处理3小时,再15°C处理1小时。将所得溶液真空浓缩,然后加入二异丙醚沉淀去保护的肽,并用10mL二异丙醚洗涤3次。将所得固体真空干燥(25°C,15Torr)至恒重。
实施例3
单环和双环胰岛素样肽的去保护。通用方法
将上述实施例1中所得带保护的RLX-A链和B链(0.05mmol)用5mLTFA/TIPS/苯甲醚/水(91∶4∶1∶4)5°C处理3小时,再15°C处理1小时。将所得溶液真空浓缩,然后加入二异丙醚沉淀去保护的肽,并用10mL二异丙醚洗涤3次。所得固体物质真空干燥(25°C,15Torr)至恒重。对于各条A链和B链重复此程序。
实施例4
纯化去保护的肽和它们的单环与双环衍生物。通用程序。
将粗制去保护的RLXIA、RLX2A、Met24(O)-RLX1B和Met25(O)-RLX2B以及单环和双环衍生物的三氟乙酸盐溶于含25%乙腈的水中,加到10x25mm的半制备型柱上。Lichrospher 100,RP-18,12微米柱(Merck);A相=含1%TFA的乙腈,B相=含1%TFA的水;线性梯度25%-A至65%-A洗脱30分钟。纯化产率为30-80%。对RLX2A、Met24(O)-RLX1B和Met25(O)-RLX2B和单环及双氧化衍生物重复此程序。
实施例5
从CTC树脂上切割并同时用碘单氧化带保护的肽。制备胰岛素样肽的单氧化A链和B链。
将上述实施例1和2所得结合于带保护肽的N-和侧链上的树脂用5mL NMP洗涤4次,用5mL IPA洗涤3次,最后用7mL DCM洗涤5次,以完全去除NMP和其它碱性组分。冷却树脂至0°C。滤除DCM后,用含相当于树脂所结合肽的10当量碘的10mL 1%TFA的DCM溶液5°C处理2次。所得混和液在0°C搅拌5分钟后过滤(代替1%TFA,可用相同体积的二氯甲烷/乙酸/三氟乙醇混和液,结果相似)。用10mL DCM洗涤树脂3次。合并的滤液加热至15°C再搅拌30分钟。然后在该滤液中加入吡啶(相当于TFA的1.3当量)以中和TFA。然后将该DCM切割液与等体积的3%硫代硫酸钠水溶液混和以去除过量的碘。混和液脱色表明碘去除。所得混和液减压蒸馏(350torr,28°C)以去除DCM。去除DCM后沉淀所得肽或肽区段。所得肽用水洗涤并在30-35°C下15Torr真空干燥。如实施例2、3和4所述进行去保护和纯化。总产率45-65%。对所有分子重复该程序。
实施例6
通过用DMSO氧化来合成带保护的单环胰岛素样肽。通用方法。
A1.Cys(Mmt)的选择性去保护。胰岛素样肽的部分去保护。
取结合于上述实施例B1-B5中所得含有2个用Trt保护的半胱氨酸残基和2个用Mmt保护的半胱氨酸残基的带保护肽(0.005mmol)的N-和侧链上的树脂用5mL NMP洗涤4次,用5mL IPA洗涤3次,最后用7mL DCM洗涤5次,以完全去除NMP和其它碱性组分。冷却树脂至0°C,过滤去除DCM,用25mL 1.5%TFA的DCM溶液5°C处理4次,所述溶液含有相当于该树脂所连接肽的10当量的三乙基硅烷。合并的滤液于15°C再搅拌2小时。然后在该滤液中加入吡啶(相当于TFA的1.3当量)以去除TFA。然后将所得DCM切割液与等体积水混和。所得混和液减压蒸馏(350torr,28°C)以去除DCM。去除DCM后沉淀在S-Mmt处选择性去保护的肽和肽区段。所得肽然后用水洗涤并在30-35°C下15Torr真空干燥。
A2.用DMSO氧化使游离半胱氨酸形成单环。
将如A1方法所述得到的肽(0.005mmol)溶于5ml DMSO中,25°C搅拌2小时。然后加入5ml水继续搅拌30分钟。然后将所沉淀的单环带保护肽用水洗涤5次并真空(30°C,15Torr)干燥至恒重。按实施例2、3和4所述进行去保护和纯化。总得率范围为50-70%。
实施例7
合成胰岛素样肽及其衍生物的双环A链。通用方法。
A1.用碘氧化胰岛素样肽及其衍生物的带保护单环A链,其中有2个半胱氨酸残基的侧链用Trt基团保护。
将a其中有2个半胱氨酸残基的侧链用Trt基团保护的胰岛素样肽及其衍生物的单环带保护A链(0.005mmol)溶于5ml DCM/TFE(7∶3)中。溶液冷却至5°C后加入含10当量碘的5ml DCM液,混和液搅拌1小时。然后将该DCM切割液与等体积3%硫代硫酸钠水溶液混和以去除过量的碘。混和液脱色表明碘去除。所得混和液减压蒸馏(350torr,28°C)以去除DCM。去除DCM后沉淀所得肽或肽区段。所得沉淀肽用水洗涤后30-35°C下15Torr真空干燥。按实施例2、3和4所述进行去保护和纯化。总得率范围为50-80%。
A2.用碘氧化二个半胱氨酸侧链用Acm基团保护的胰岛素样肽及其衍生物的带保护单环A链。
将其中有2个半胱氨酸残基的侧链用Acm基团保护的胰岛素样肽及其衍生物的单环带保护A链(0.005mmol)溶于5ml AcOH/TFE(5∶5)中。溶液冷却至5°C后加入含20当量碘的5ml TFE液,混和液搅拌1小时。然后将该DCM切割液与5倍体积的3%硫代硫酸钠与抗坏血酸的水溶液混和以去除过量的碘。混和液脱色表明碘去除。所得混和液减压蒸馏(350torr,28°C)以去除DCM。去除DCM后沉淀所得肽或肽区段,然后用水洗涤,在30-35°C下15Torr真空干燥。按实施例2、3和4所述进行去保护和纯化。总得率范围为50-60%。
A3.用DMSO氧化胰岛素样肽及其衍生物的去保护单环A链,通用方法。
将胰岛素样肽及其衍生物的单环去保护A链(0.005mmol)溶于4ml pH=4的乙酸铵缓冲液中。然后加入1ml DMSO,混和液在15°C搅拌24小时。按实施例4所述从所得溶液分离并纯化双环肽。总得率范围为65-85%。
A4.用DMSO氧化胰岛素样肽及其衍生物的直链去保护单环A链,通用方法。
将胰岛素样肽及其衍生物的直链去保护单环A链(0.005mmol)溶于4ml pH=4的乙酸铵缓冲液中。然后加入1ml DMSO,混和液在15°C搅拌24小时。按实施例4所述从所得溶液分离并纯化双环肽。总得率范围为60-80%。
实施例8
合成胰岛素样肽及其衍生物的单环B链。通用方法。
将胰岛素样肽及其衍生物的直链去保护B链(0.005mmol)溶于4ml pH=10.5的甘氨酸钠缓冲液中。然后加入1ml DMSO,混和液在15°C搅拌24小时。按实施例4所述从所得溶液分离并净化环肽。三次实验的平均得率为25-45%。
实施例9
通过线性结合胰岛素样肽的A链与胰岛素样肽及其衍生物的直链B链合成胰岛素样肽及其衍生物,通用方法。
将去保护的胰岛素样肽直链A链(0.006mmol)与胰岛素样肽的直链B链(0.005mmol)溶于4ml pH=10.5的甘氨酸钠/6-N盐酸胍(4∶1)缓冲液中。然后在12小时内加入1ml DMSO,混和液在15°C搅拌4小时。按实施例4所述从所得溶液分离并纯化胰岛素样肽。三次实验胰岛素样肽的平均得率为15-35%。
实施例10
通过胰岛素样肽的直链A链与胰岛素样肽及其衍生物的环形B链的线性组合合成胰岛素样肽及其衍生物,通用方法
将胰岛素样肽的直链去保护A链(0.005mmol)与胰岛素B链或衍生物的环形肽(0.005mmol)溶于4ml pH=10.5的甘氨酸盐/6-N盐酸胍(4∶1)缓冲液中。然后在12小时内加入1ml DMSO,混和液在15°C搅拌4小时。从所得溶液中按实施例4所述通过浸蚀(etching)分离胰岛素样肽。三次实验胰岛素样肽的平均得率按所用的B链计算为5-70%。
实施例11
合成胰岛素衍生肽,及其在胰岛素B肽和衍生物的直链中结合胰岛素样肽及其衍生物的单环A链,通用方法
将胰岛素样肽A链肽的去保护单环或产生子(0.006mmol)与胰岛素样肽的环形B链肽或产生子(0.005mmol)溶于4ml pH=10.5的甘氨酸盐/6-N盐酸胍(4∶1)缓冲盐液中。然后在12小时内逐步加入1ml DMSO,混和液在15°C搅拌4小时。从所得溶液中按实施例4所述通过浸蚀分离胰岛素样肽。三次实验胰岛素样肽的平均得率按所用的B链计算为12-36%。
实施例12
通过结合胰岛素样肽的单环A链与胰岛素样肽及其衍生物的直链B链合成胰岛素样肽及其衍生物,通用方法
将胰岛素样肽或其衍生物的去保护单环A链(0.006mmol)与胰岛素样肽或其衍生物的环形B链(0.005mmol)溶于4ml pH=10.5的甘氨酸钠/6-N盐酸胍(4∶1)缓冲液中。然后在12小时内逐步加入1ml DMSO,混和液在15°C搅拌4小时。从所得溶液中按实施例4所述分离并纯化胰岛素样肽。三次实验胰岛素样肽的平均得率按所用的B链计算为10-40%。
实施例13
通过线性结合胰岛素样肽及其衍生物的双环A链与胰岛素样肽及其衍生物的直链B链合成胰岛素样肽及其衍生物,通用方法
将胰岛素样肽或其衍生物的去保护双环A链(0.006mmol)与胰岛素样肽或其衍生物的直链B链(0.005mmol)溶于4ml pH=10.5的甘氨酸钠/6-N盐酸胍(4∶1)缓冲液中。然后在12小时内逐步加入1ml DMSO,混和液在15°C搅拌4小时。从所得溶液中按实施例4所述分离并纯化胰岛素样肽。三次实验胰岛素样肽的平均得率按所用的B链计算为5-80%。
实施例14
通过结合胰岛素样肽及其衍生物的双环A链与胰岛素样肽及其衍生物的环形B链合成胰岛素样肽及其衍生物,通用方法
将胰岛素样肽或其衍生物的去保护双环A链(0.011mmol)与胰岛素样肽或其衍生物的环形B链(0.01mmol)溶于15ml pH=10.5的甘氨酸钠/6-N盐酸胍(4∶1)缓冲液中。然后5-10°C搅拌下,用时48加入5ml的0.4mmol二硫苏糖醇水溶液。从所得溶液中按实施例4所述分离并纯化胰岛素样肽。三次实验胰岛素样肽的平均得率按所用的B链计算为20-75%。
附图说明
图1.胰岛素样肽的一级结构。阴影表示如图2示意图所示连接在一起的半胱氨酸残基。A链N-末端的第1半胱氨酸与A链N-末端第3半胱氨酸连接。A链N-末端的第2半胱氨酸与B链N-末端第1半胱氨酸连接。A链N-末端的第4半胱氨酸与B链N-末端第2半胱氨酸连接。
图2.胰岛素样肽的示意图。粗线代表肽链。S代表此肽中半胱氨酸残基的硫原子,细线代表化学键。
图3.随机结合直链A链与直链B链制备胰岛素样肽的示意图。粗线代表肽链。S代表此肽中半胱氨酸残基的硫原子,细线代表化学键。
图4.随机结合单环A链与直链B链制备胰岛素样肽的示意图。粗线代表肽链。S代表此肽中半胱氨酸残基的硫原子,细线代表化学键。
图5.随机结合双环A链与直链B链制备胰岛素样肽的示意图。粗线代表肽链。S代表此肽中半胱氨酸残基的硫原子,细线代表化学键。
图6.随机结合双环A链与环形B链制备胰岛素样肽的示意图。粗线代表肽链。S代表此肽中半胱氨酸残基的硫原子,细线代表化学键。
图7.随机结合直链A链与环形B链制备胰岛素样肽的示意图。粗线代表肽链。S代表此肽半胱氨酸残基的硫原子,细线代表化学键。
图8.随机结合单环A链与环形B链制备胰岛素样肽的示意图。粗线代表肽链。S代表此肽中半胱氨酸残基的硫原子,细线代表化学键。
图9.结合含有至少1个半胱氨酸残基的双环肽的示意图。此反应中可形成多达4种异构体。粗线代表肽链。S代表此肽中半胱氨酸残基的硫原子,细线代表化学键。
Claims (21)
1.一种制备由二条不同的肽链:A链与B链组成的肽(图1)及其衍生物的方法,其中所述二条链结合在一起,所述方法采用
i)在还原性化合物存在下,使含有至少2个二硫键的A链与含有至少1个二硫键的B链反应,优选包含A链和B链的硫醇,A链和B链为含有游离巯基;
ii)在温和的氧化剂存在下,使含有至少2个二硫键的A链与含有至少2个半胱氨酸残基的B链反应,所述氧化剂优选DMSO、空气或过氧化氢溶液;
iii)在温和的氧化剂存在下,使含有至少1个二硫键和至少2个带游离巯基的半胱氨酸残基的A链与含有至少1个二硫键的B链反应,所述氧化剂优选DMSO、空气或过氧化氢溶液;
iv)在温和的氧化剂存在下,使含有至少1个二硫键和至少2个带游离巯基的半胱氨酸残基的A链与含有至少1个二硫键的B链反应,所述氧化剂优选DMSO、空气或过氧化氢溶液。
2.如权利要求1所述的制备由二条不同肽链:A链与B链组成的肽的方法,其特征在于,在适于形成至少1个分子间二硫键的条件下将这二条链结合在一起,A链和B链中的至少一条另含有至少1个分子内二硫键。
3.如权利要求1和2所述的方法,其特征在于,所述A链和B链中至少其一是胰岛素样肽的A链或B链。
4.如权利要求1-3所述的方法,其特征在于,所述胰岛素样肽的链如图1所示人来源胰岛素样肽那样是全链、链片段、经修饰的链,是已用一个或多个天然和非天然氨基酸延伸的人或动物来源的胰岛素样肽链,是人或动物来源的胰岛素(INS)的A链或B链,松弛素1(RLN1)的A链或B链,松弛素2(RLN2)的A链或B链,对应于***1(IGF-1)A链或B链的区域,和对应于***2(IGF-2)A链或B链的区域。
5.一种如权利要求1-4所述制备嵌合肽的方法,包括将树脂上或三苯甲基或二苯甲基类接头上的天然或非天然氨基酸的衍生物酯化,使之在固相上依次与胰岛素样肽的可选带保护的残基反应。
6.如权利要求1-4中任一项所定义并用权利要求1所述方法制备并为药学活性的合成胰岛素样多肽,或其药学上可接受的盐、衍生物和前体或药物制剂。
7.权利要求6所述的多肽,其特征在于,用于治疗胶原蛋白产生相关疾病。
8.如权利要求6所述的多肽,其特征在于,用于心脏保护,动脉粥样硬化症,心脏、肾脏、动脉、肝脏和胰腺的纤维化症。
9.如权利要求6所述的多肽,其特征在于,用于所有类型的子痫。
10.如权利要求6所述的多肽,其特征在于,用于抵御关节炎和所有类型的疼痛。
11.如权利要求6所述的多肽,其特征在于,用于抵御衰老。
12.如权利要求6所述的多肽,其特征在于,用于代谢疾病,如所有类型的糖尿病和肥胖。
13.如权利要求6所述的多肽,其特征在于,用作生长因子。
14.一种包含权利要求6-13中任一项所述多肽的药物制剂,采用药学上可接受的运载体给药。
15.一种含7-35个氨基酸序列的双环多肽,其中至少4个氨基酸是半胱氨酸残基,所述半胱氨酸残基通过2个二硫键连接在一起,并且包括典型序列CysCysXYZCys,其中X、Y和Z是天然或非天然氨基酸。
16.如权利要求15所述的多肽,其属于胰岛素样肽类别,小于或大于胰岛素样肽,或是它们的衍生物。
17.如权利要求15-16所述多肽在制备由二条不同的肽链:A链与B链组成的肽中的应用,其特征在于,所述A链或B链通过至少一个分子间二硫键连接在一起,这二条链中至少一条另含一个分子内二硫键。
18.如权利要求15-16所述的多肽,其特征在于,其作为药物给予,并如权利要求17所述,在人或动物体内与可能在人或动物体内沉淀的天然蛋白或非天然蛋白的寡聚体、凝聚蛋白的斑块、天然或修饰蛋白的凝聚体、突变蛋白质或折叠不当(错误折叠)的蛋白质反应。
19.如权利要求15-16所述的多肽,其特征在于,用于天然蛋白错误折叠引起的构象疾病,如衰老、阿尔茨海默症、帕金森症、糖尿病和某些类型的癌症。
20.如权利要求15-16所述的多肽,其特征在于,在人或动物体内用作氧化酶,或用于生物技术制备蛋白质的正确氧化。
21.一种包含如权利要求15-16中任一项所定义多肽的药物制剂,并采用药学上可接受的运载体给药。
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CN104892748A (zh) * | 2014-03-04 | 2015-09-09 | 深圳翰宇药业股份有限公司 | 人类松弛素-2衍生物以及由其制备人类松弛素-2的方法 |
CN105884879A (zh) * | 2015-01-26 | 2016-08-24 | 漳州博欣生物技术有限公司 | 一种胰岛素类似物的化学合成方法 |
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JP6276690B2 (ja) * | 2011-08-04 | 2018-02-07 | ファリス バイオテック ゲーエムベーハー | ヒトリラキシン−2を調製する方法 |
US20160031962A1 (en) * | 2012-04-20 | 2016-02-04 | Kleomenis K. Barlos | Solid phase peptide synthesis of insulin using side chain achored lysine |
AU2017322552B2 (en) | 2016-09-06 | 2021-12-02 | Chemical & Biopharmaceutical Laboratories Of Patras S.A. | Proinsulin derivatives |
ES2963839T3 (es) | 2017-02-08 | 2024-04-02 | Bristol Myers Squibb Co | Polipéptidos de relaxina modificados que comprenden un potenciador farmacocinético y usos de los mismos |
JP2020138975A (ja) * | 2020-05-18 | 2020-09-03 | ケミカル アンド バイオファーマシューティカル ラボラトリーズ オブ パトラ エス.エー. | 側鎖結合を介する固相ペプチド合成 |
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CN105481974A (zh) | 2016-04-13 |
HK1220701A1 (zh) | 2017-05-12 |
CA2776995C (en) | 2019-04-09 |
US20120302498A1 (en) | 2012-11-29 |
CN102844328B (zh) | 2016-01-20 |
US10711053B2 (en) | 2020-07-14 |
WO2011042762A2 (en) | 2011-04-14 |
US20160009778A1 (en) | 2016-01-14 |
CA2776995A1 (en) | 2011-04-14 |
EP2486045B1 (en) | 2018-08-22 |
GR1007010B (el) | 2010-10-07 |
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EP2486045A2 (en) | 2012-08-15 |
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